JP2767124B2 - Electrodes for analysis / test / measurement and enzyme sensors using them - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an electrode having high sensitivity, a wide range of use, easy size reduction, and excellent biocompatibility, and an enzyme sensor using the electrode.

[Prior Art and Problems to be Solved by the Invention] Conventionally, an enzyme sensor that detects an enzymatic reaction by transferring electrons between a membrane on which an enzyme is immobilized and an electrode has been studied.

However, metals and carbon materials are conventionally used as electrodes for measuring electrochemical and biochemical reactions in enzyme sensors, but they are suitable for corrosion resistance, stability, biocompatibility, miniaturization, etc. There is nothing.

In addition, conventional enzyme sensors generally have low sensitivity and poor stability.

Specifically, GOD (glucose oxidase)
Enzyme sensors that fix oxygen and monitor the consumed oxygen or the generated hydrogen peroxide have many problems such as low sensitivity, poor stability, and limited use.

Utilize the phenomenon that pH changes by enzymatic reaction
FET biosensors have the drawback that not only are they low in sensitivity, but only those that release H ⁺ can be used.

Meanwhile, as an electrode for electrochemical testing and analysis focusing on the characteristics of diamond, an electrode made of diamond added with conductivity by ion implantation is known (Japanese Patent Application Laid-Open No. 58-160860).

However, this electrode, which modifies the surface of diamond to provide conductivity, has poor homogeneity in the conductivity of the electrode, has low accuracy, and has difficulty in extracting current.

[Object of the Invention] An object of the present invention is to provide various kinds of electrochemical analysis, testing, and measurement including enzyme sensors having high sensitivity, a wide range of use, easy current extraction, and excellent biocompatibility. An electrode and an enzyme sensor using the same.

[Means for Achieving the Object] The enzyme sensor of the present invention is an electrode for analysis / test / measurement made of conductive diamond containing impurities formed on a substrate by a gas phase method. An enzyme sensor characterized in that an impurity-containing conductive diamond electrode formed on a substrate is coated with an enzyme-containing conductive resin layer.

 Hereinafter, the present invention will be described in detail.

-Substrate-As a substrate in the electrode or the enzyme sensor,
There is no particular limitation as long as it has insulation properties and heat resistance and can form a diamond layer on its surface, and it can be appropriately selected according to the purpose.

Examples of the substrate include oxides, nitrides and carbides of metals such as silicon, manganese, vanadium, thallium, aluminum, titanium, tungsten, molybdenum, germanium, and chromium, Al ₂ O ₃ —Fe, and TiC—Ni.
System, may be mentioned TiC-Co based and B ₄ C-Fe system or the like of the cermet and various ceramics.

Among them, SiO ₂ is preferable.

These substrates may be subjected to a surface treatment or a coating treatment for further improving the adhesion to a diamond electrode formed by a vapor phase synthesis method. An intermediate layer may be provided, and by appropriately performing such a treatment, the adhesion between the diamond electrode and the substrate can be further improved.

Although there is no particular limitation on the shape of the substrate, it is usually a flat plate.

-Impurity-conducting conductive diamond electrode-The electrode or enzyme sensor of the present invention has an impurity-conducting conductive diamond electrode.

The impurity-containing conductive diamond electrode contains impurities doped in the diamond layer.

As the impurities, for example, B, Al, Ga, In and
Examples include Group IIIb elements of the periodic table such as Tl and Group Vb elements of the periodic table such as N, P, Sb, and Bi.

 Among these, B is preferable.

The thickness of the impurity-containing conductive diamond electrode is
Usually, it is 1,000 to 10 μm, especially 2,000 to 5,000 mm.

-Formation of impurity-containing conductive diamond layer-The impurity-containing conductive diamond layer can be formed as follows.

That is, a gas obtained by exciting a carbon source gas containing a simple substance of a group IIIb element or a group Vb element and / or at least one of its compounds is brought into contact with a substrate.

The simple substance of the group IIIb element or the group Vb element of the periodic table and at least one compound thereof contained in the carbon source gas to be used are usually used as a dopant gas.

The simple substance of the group IIIb element or the group Vb element is as described above.

Examples of such compounds include B, Al, G
_{a, In, Tl, B 2} H 6, B 4 H 10, CH 3 BCl 2, [(CH 3) AlBr 2]
₂ , CH ₃ ) ₂ Al Cl, [(CH ₃ ) ₃ Al] ₂ , Ga ₂ H ₆ , (CH ₃ ) ₃
Ga, (CH ₃ ) ₂ GaBH ₄ , (CH ₃ ) ₃ In, (CH ₃ ) ₃ Tl, N ₂ , N
_{H 3, PH 3, AsH 3} , N 2 H 4, CH 3 AsBr 2, CH 3 AsH 2, (CH 3) 3 A
s, (CH ₃ ) ₃ Bi, SbH ₃ , ClCH ₂ SbCl, CH ₃ SbH ₂ , (CF ₃ ) ₃ S
b and the like.

The simple substance of the group IIIb element, the simple substance of the group Vb element and the compound thereof may be used alone or in combination of two or more.

The carbon source gas is a simple substance of the group IIIb element and / or
Alternatively, a carbon raw material is contained together with at least one of the compounds.

As the carbon raw material, gases such as various hydrocarbons, halogen-containing compounds, oxygen-containing compounds, and nitrogen-containing compounds can be used.

Examples of the hydrocarbon compound include paraffinic hydrocarbons such as methane, ethane, propane, and butane; ethylene,
Olefin hydrocarbons such as propylene and butylene; acetylene hydrocarbons such as acetylene and allylene; diolefin hydrocarbons such as butadiene; alicyclic hydrocarbons such as cyclopropane, cyclobutane, cyclopentane and cyclohexane; cyclobutadiene, benzene , Toluene, xylene, aromatic hydrocarbons such as naphthalene; methyl chloride,
Examples thereof include halogenated hydrocarbons such as methyl bromide, methylene chloride, and carbon tetrachloride.

Examples of the oxygen-containing compound include ketones such as acetone, diethyl ketone and benzophenone; alcohols such as methanol, ethanol, propanol and butanol;
Ethers of methyl ether, ethyl ether, ethyl methyl ether, methyl propyl ether, ethyl propyl ether, phenol ether, acetal, cyclic ethers (dioxane, ethylene oxide, etc.); acetone, pinacolin, methyl oxide, aromatic ketones (acetophenone, benzophenone) Ketones such as diketone and cyclic ketone; aldehydes such as formaldehyde, acetaldehyde, butyraldehyde, and benzaldehyde; organic acids such as formic acid, acetic acid, propionic acid, succinic acid, butyric acid, oxalic acid, tartaric acid, and stearic acid; Acid esters such as methyl acetate and ethyl acetate; dihydric alcohols such as ethylene glycol and diethylene glycol;
Examples thereof include carbon monoxide and carbon dioxide.

Examples of the nitrogen-containing compound include, for example, amines such as trimethylamine and triethylamine.

As the carbon raw material, although not a simple substance, a fourth class dangerous substance defined by the Fire Services Act; a first petroleum such as gasoline; a second petroleum such as kerosene, turpentine oil, cerebral oil, and pine oil; Gases such as third petroleum such as heavy oil and fourth petroleum such as gear oil and cylinder oil can also be used. Further, the above-mentioned various carbon compounds may be used as a mixture.

Among these carbon raw materials, methane, ethane, paraffinic hydrocarbons such as propane, ethylene, olefinic hydrocarbons such as propylene, or ketones such as acetone, benzophenone, etc.
Preferred are alcohols such as methanol and ethanol, and oxygen-containing compounds such as carbon monoxide and carbon dioxide gas.

If desired, a carrier gas such as a hydrogen gas or an inert gas can be used together with the carbon raw material.

In the carbon source gas, the content of the simple substance of the group IIIb element and the simple substance of the group Vb element and at least one of the compounds thereof (hereinafter, these may be referred to as dopant elements) is determined by the ratio of the dopant element to the dopant element. The ratio [(dopant element) / (carbon raw material) molar ratio] to the carbon raw material is usually 10 ^-8 to 10 ^-1 , preferably 10 ^-7 to 10 ^-1 . If the ratio is less than 10 ^-8 , an impurity-containing conductive diamond layer having sufficient conductivity may not be formed.

On the other hand, when it exceeds 10 ^-1 , a uniform and good electrode film as diamonds cannot be obtained, and for example, application as an electrode for an enzyme sensor may be difficult.

In the present invention, as shown in FIG. 1, a gas obtained by exciting the carbon source gas is brought into contact with a substrate 1 to form an impurity-containing conductive diamond layer 2 on the substrate.

The means for exciting the carbon source gas is not particularly limited as long as it is a method capable of forming crystalline diamond (which may contain amorphous carbon) or diamond-like carbon by a gas phase method. For example, a method of plasma decomposition by DC or AC arc discharge, a method of plasma decomposition by high frequency dielectric discharge, a method of plasma decomposition by microwave discharge (including magnetic field-CVD method), a method of decomposition by light energy, or a method of plasma decomposition Using an ion chamber or an ion gun to extract ions by an electric field, a pyrolysis method (including EACVD method) that thermally decomposes by heating with a hot filament, a combustion flame method, and a sputtering method. Can be adopted.

In the method of the present invention, the reaction usually proceeds under the following conditions to form an impurity-containing conductive diamond layer on the substrate.

That is, since the temperature of the surface of the base varies depending on the means for exciting the carbon source gas, it cannot be unconditionally determined, but is usually from room temperature to 1,200 ° C., preferably from room temperature to 1,200 ° C.
-1,100 ° C.

If the above-mentioned temperature is lower than room temperature, the deposition rate of the diamond layer containing impurities is reduced, or the homogeneity of the precipitate is lost. On the other hand, if the temperature is higher than 1,200 ° C., the effect corresponding thereto is not exhibited, and it is disadvantageous in terms of energy efficiency, and the formed impurity-containing diamond layer may be etched.

The reaction pressure is usually 10 ^-6 to 10 ³ torr, preferably 10 ^-5 t
orr to 800 torr.

If the reaction pressure is lower than 10 ^-6 torr, the film formation rate of the impurity-containing diamond layer may be reduced, or the impurity-containing diamond layer may not be deposited.

On the other hand, if it is higher than 10 ³ torr, the corresponding effect may not be obtained.

The total flow rate of the raw material gas including the carbon source gas is usually 1 to 1,000 SCCM, preferably 10 to 500 SCCM.

The reaction time cannot be determined unequivocally because it varies depending on the temperature of the surface of the substrate, the reaction pressure, the required thickness of the diamond layer containing impurities, and the like, but it can usually be within 10 hours.

-Formation of Impurity-Containing Conductive Diamonds Electrode-After the impurity-containing conductive diamonds film 2 is laminated on the base 1, masking is performed and etching is performed, as shown in FIG. Then, the conductive diamond electrode 3 mixed with impurities is formed.

The etching treatment is performed by placing a mask on the surface of the impurity-containing conductive diamond layer, and bringing a gas obtained by exciting an oxygen-containing etching gas into contact with the impurity-containing conductive diamond layer.

The oxygen-containing etching gas is not particularly limited as long as it contains an oxygen atom. For example, oxygen-containing gas such as oxygen, air, carbon monoxide, carbon dioxide, water, hydrogen peroxide, various nitrogen oxides, etc. In addition, a mixed gas with another gas such as a hydrogen gas, a halogen gas, a nitrogen gas, and an argon gas may be used. Among these oxygen-containing gases, oxygen gas and air-containing gas are preferable.

Exciting means of the oxygen-containing etching gas, high-frequency plasma discharge method, RF plasma discharge method, DC plasma discharge method,
A microwave plasma discharge method or the like (including a magnetic field discharge method) can be suitably employed.

As the supply amount of the etching gas to the reaction vessel,
Usually, it is 1 to 1,000 SCCM, preferably 10 to 500 SCCM.

If the supply amount is less than 1 SCCM, the etching rate may decrease.If the supply amount exceeds 1,000 SCCM, for example, when microwave discharge is employed as an etching gas excitation means, the discharge is stable. May not work.

The temperature of the surface of the impurity-containing conductive diamond layer is usually from room temperature to 1,200 ° C.

The reaction pressure is usually 10 ^-6 to 10 ³ torr, preferably 10 ^-5
~ 800 torr.

If the reaction pressure is less than 10 ^-6 torr, the etching rate may decrease, and if it exceeds 10 ³ torr, the corresponding effect may not be obtained.

The time required for the etching treatment depends on the film thickness and the reaction conditions of the impurity-containing conductive diamond layer, and thus cannot be specified unconditionally.
It can be about 5 minutes to 2 hours.

By contacting the impurity-containing conductive diamond layer with the excitation etching gas as described above, an impurity-containing conductive diamond electrode is formed.-Enzyme-containing conductive resin layer- An enzyme is immobilized on a conductive resin such as polypyrrole, ferrocene polymer, polythiophene, polyfuran, polyaniline, and polyacetylene.

The conductive resin can be manufactured by, for example, electrolytic polymerization, oxidative polymerization, or the like.

As the enzyme, various enzymes can be selected by a biochemical reaction.For example, a flavin-based enzyme, a heme-based enzyme, or the like can be used.For example, when the enzyme acts as a catalyst in the oxidation reaction of NADH, diaphorase is suitable. It is.

The fixed amount of the enzyme cannot be determined unconditionally depending on the type of biochemical reaction, the type of enzyme, and the like, but is generally preferably 0.01 to 10% based on the weight of the conductive resin.

Also, the thickness of the conductive resin layer is not particularly limited, but usually, 0.1 to 100 μm is appropriate.

As shown in FIG. 3, the enzyme sensor of the present invention comprises an impurity-containing conductive diamond electrode 3 formed on a substrate 1 by a vapor phase method and covered with an enzyme-containing conductive resin layer 4. If a voltage is applied between the mixed conductive diamond electrodes, a current flows due to the progress of the enzyme reaction. Therefore, by detecting this current, it is possible to detect the type of the enzyme reaction, the reaction speed, and the like.

Example First, an SiO ₂ substrate was placed in a reaction chamber of a microwave plasma CVD apparatus.

Next, 7 scc of CO gas was supplied as a raw material gas into the reaction chamber.
m, hydrogen gas 93 sccm, B gas (use B ₂ H ₆ , H ₂ ) 195
ppm, the pressure in the reaction chamber was set to 40 Torr, and the temperature of the SiO ₂
350W from 2.45GHz microwave power supply under the condition of 00 ℃
Set to.

When plasma treatment was performed under these conditions for 30 minutes, SiO ₂
A B-containing conductive diamond film having a thickness of 3,000 mm was formed on the substrate.

Next, masking was performed and an etching treatment with O ₂ was performed to form a diamond electrode having a width 1 of 5 μm.

Furthermore, a polypyrrole film to which diaphorase was added was coated on the SiO ₂ substrate including this electrode, and finally, a predetermined wiring was provided.

The obtained enzyme sensor was placed in a substrate containing NADH, and evaluated as an enzyme switch.
The switch was turned off when NAD ⁺ was turned to NAD ⁺ by the enzyme in the polypyrrole film (ON during steady state).

Although an example of an enzyme sensor using a diamond electrode has been described above, the electrode of the present invention can be widely used as an electrode for electrochemical analysis / test / measurement due to its excellent characteristics.

[Effects of the Invention] The electrode of the present invention is an impurity-conducting conductive diamond electrode formed by a gas phase method and having excellent homogeneity, and the enzyme sensor uses an impurity-conducting conductive diamond electrode formed by a gas phase method. Since the enzyme-containing conductive resin layer is laminated via the intermediary layer, the sensitivity is high, the range of use is wide, the size is easily reduced, and the biocompatibility is excellent. Therefore, it greatly contributes to the medical measurement field.

[Brief description of the drawings]

1 to 3 show steps for obtaining the enzyme sensor of the present invention. DESCRIPTION OF SYMBOLS 1 ... Base, 2 ... Impurity-conducting conductive diamond layer, 3 ... Impurity-conducting conductive diamond electrode, 4 ...
... Enzyme-containing conductive resin layer.

Claims (2)

(57) [Claims] An electrode for analysis / test / measurement comprising conductive diamond containing impurities formed on a substrate by a gas phase method. 2. An enzyme sensor comprising an impurity-containing conductive diamond electrode formed on a substrate by a vapor phase method and coated with an enzyme-containing conductive resin layer.